The bile salt export pump (BSEP, ABCB11) is an ABC transporter located primarily in the human liver. Gene regulation of BSEP is controlled by several transcription factors, including the farnesoid x receptor (FXR), which induces BSEP expression when activated by bile acids. FXR activity and its transactivation of target genes represent a complex system for regulating bile acid levels. The tightly monitored flow of bile acid through the body is referred to as enterohepatic circulation; disruption of this flow can result in the hepatotoxic condition, cholestasis. The most commonly investigated cause of cholestasis is through inhibition of BSEP, which there have been many drugs reported. BSEP inhibition is undoubtedly important, however, another key factor typically overlooked when studying a drug's effect on BSEP is its influence on BSEP gene expression, in particular, repression. The overall objective of this research project is to investigate the role and mechanism(s) of BSEP repression in the development of drug-induced cholestasis. Our hypothesis is that drugs determined to repress BSEP expression will further compound the inhibitory effects of that drug, resulting in more severe clinical consequences than for BSEP inhibitors alone. Currently our results indicate only a few potent BSEP inhibitors (BSEP IC50 values = 25 M) are also strong repressor of BSEP. These drugs were also determined to have more severe clinical implications than BSEP inhibitors alone, supporting our hypothesis that identifying drugs which both inhibit and repress BSEP will enhance the prediction of their cholestatic potential. In specific aim 1, the expression profile of BSEP, and its transcriptional regulators will be assessed in human primary hepatocytes upon treatment with select BSEP inhibitors. Functional activity of BSEP will also be monitored in order to determine the cholestatic potential of these drugs. A mechanistic study will be performed in specific aim 2 on the popular anti-diabetic drug metformin, which was found to repress BSEP expression in a concentration-dependent manner in preliminary experiments. The transcriptional activity of key regulators, as well as AMPK, will be investigated. The contribution of genetic polymorphisms on drug-induced cholestasis will be explored in specific aim 3. The frequency of the V444A mutation and cholestasis, as determined by a functional assay, will be studied in homozygous wild-type or polymorphic liver donors. Understanding that both inhibition and repression of BSEP can lead to serious clinical consequences can provide important criteria to be considered in the new drug application process. By elucidating the mechanism responsible for repression of BSEP expression, assays can be developed to screen for drugs that are both BSEP inhibitors and repressors. With the future of health care moving toward personalized medicine, based on a patient's genotype, characterizing the V444A polymorphism in the development of drug-induced cholestasis will provide valuable information to consider when patients have this susceptibility factor.